Project Summary/Abstract The goals of the proposed research are to develop functional autologous trilayered heart valve leaflets with collagen fibril orientations of a native leaflet using trilayered nanofibrous substrates and to extend this approach in developing fully autologous heart valves with native heart valve functionality. The proposed work will develop a technology to fabricate trilayered nanofibrous substrates from a FDA approved polymer mimicking trilayered structure and orientations of collagen fibrils of native heart valve leaflets. The proposed work will then apply leaflet-shaped trilayered nanofibrous substrates to develop non-contractile autologous valve leaflets mimicking the structure of native leaflets by in-body tissue engineering. The leaflet constructs will be tested in-vitro to verify their morphological, structural, and functional properties and contractility. The proposed work will then develop heart valve-shaped nanofibrous substrates containing leaflet-shaped trilayered nanofibrous substrates and circumferentially oriented tubular nanofibrous substrates to engineer autologous non-contractile heart valves with comparable properties of native heart valves through in-body tissue engineering. The engineered valves will be tested for their morphological, structural, mechanical and functional properties in-vitro. The engineered autologous valves will also be tested for clinically-relevant outcomes including function, thrombus formation, and calcification in an ovine implantation model. These valves are expected to be an important step in the development toward clinical translation. The proposed research focuses the candidate's research in a novel direction to provide training on new skills required to begin the transition to independence. The candidate holds a Ph.D. in Materials Science and Engineering from the University of Washington and is currently a research associate at Mayo Clinic. His Ph.D. thesis work involved development of biomaterials for tissue engineering and regenerative medicine. This led to his postdoctoral work that involves design and development of nanofibrous biomaterials for biological cardiac valve development. His postdoctoral work also includes development of decellularized heart valve, pericardium tissue-based heart valve and stent graft, and their functionality testing in an ovine/porcine implantation model. The candidate's immediate career goal is to transition from mentored to independent research by completing his postdoctoral training and beginning a tenure track faculty position at a major research university. This will require focusing his current projects into a novel research direction while also receiving additional training needed to successfully complete the current and future projects in cardiovascular tissue engineering as an independent investigator. The K99/R00 mechanism is the ideal means of achieving this goal. The candidate's long-term career objective is to establish an independent and extramurally funded translational research program within the field of cardiovascular tissue engineering that will meaningfully improve patient care and train the next generation of scientists, physicians, and engineers. Research career development during the award will include working with an interdisciplinary mentoring team of clinicians, scientists, and engineers. The candidate's primary mentor, Dr. Amir Lerman, M.D., is the chair of Cardiovascular Research at Mayo Clinic and provides expertise in cardiovascular biology and clinical are Dr. Leigh Griffith, Ph.D., who is a professor of cardiovascular diseases at Mayo Clinic and provides expertise in biomaterials and in-vivo recipient inflammatory, immune and regenerative responses in cardiovascular area, Dr. John Stulak, M.D., is a professor of cardiovascular surgery at Mayo Clinic and provides expertise in surgical treatment of advanced heart failure, cardiology. The candidate's co-mentors and Dr. Robert Tranquillo, Ph.D., chair of the Department of Biomedical Engineering at the University of Minnesota, provides expertise in biomedical engineering and cardiovascular tissue engineering. Working with his mentors, the candidate will train in scaffold and mold design, cardiovascular physiology, cell biology and pathology, all aspects of in-body tissue engineering in ovine model, functionality tests of tissue-engineered valves and ovine model analysis of novel cardiac valves. The candidate will also train in other essential skills including communication of research findings, mentoring, and project management. Finally, educational opportunities such graduate coursework in molecular cell biology , cardiovascular physiology as well as various research and clinical seminar series, will round out the training experience. Mayo Clinic offers a variety of educational and support services through the Graduate School, College of Medicine, Office of Research Education, and Center for Clinical and Translation Science that will facilitate the necessary training. Mayo Clinic is committed to supporting translational research and recently established the Center for Regenerative Medicine as a strategic initiative. World experts in a variety of fields are available for collaboration with the common goal to improve patient care. Mayo also offers a variety of research resources and facilities including core facilities such as the Microscopy and Cell Analysis Core, the Biostatistics Core, the Histology Core, and the Materials and Structural Testing Core. The Division of Engineering features a full machine shop, electrical shop, and glassblowing shop to support research requests for engineering design and development. Mayo also has several animal facilities including the Cardiovascular Innovation Laboratory, which features a full cardiac catheterization laboratory dedicated to animal studies.